Compendial Requirements for Automated Microbiological Method Validation

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PDA Letter • May 2006

Pharmacopeial microbiological

tests detect and/or enumeratemicrobes that replicate in thepresence of microbiological media.The challenge presented by adopt-ing modern rapid microbiologicalmethods is determining whether ornot they represent the automationof a current compendial method orif they represent an “alternative” toa compendial method. The answerimpacts how a firm should qualifyand validate the new rapid methodand may provide an opportuntiyfor a more streamlined validationprotocol.

The difference is critical becauseof two chapters in the U.S.Pharmacopeia. USP Chapter<16> “Automated Methods of

Analysis” has been very useful tothe chemistry community since itsintroduction in 1975. It provides ameans to qualify new automation

methods without engenderingthe full burden of a completequalification/validation process, asdescribed in USP Chapter <1225>“Validation of Alternative Methods.”Chapter <16> provides examplesof several tests that are amenableto automation in the chemistrylaboratory, but does not addressmicrobiological methods. A secondconsideration in the method valida-tion is instrument qualification.

Although a general GMP require-ment, instrument qualificationstudies are not addressed in thesechapters, a failing USP is address-ing through the recently proposed<1058> “Analytical InstrumentQualification.”1

With interest in rapid microbio-logical methods rising, there isa variety of new technologiesavailable to the quality control

(QC) microbiology laboratory that

will move the microbiology labinto the 21st century. While manyof the more widely discussed rapidmethods are based on technolo-gies completely dissimilar to thecurrent pharmacopeial methods(PCR, viable dye, flow cytometry,etc.), several automated micro-biological tests rely on traditionalmicrobiological methodologies todetect and count microorganisms.There are several rapid microsystems currently available thatautomate detection and enumera-tion of cells replicating to formcolonies on plates containingnutrient media. Examples of thesetechnologies are the QCount fromSpiral Biotech, the ProtoCol fromMicroBiology International (MI)and the Growth Direct™ System from Genomic Profiling Systems(GPS).

The regional compendia aremoving forward on the questionof rapid microbiological methods.The USP draft chapter <1223>“Validation of Alternative Micro-biological Methods”2 will be officialin August of this year, and the EPchapter 5.1.6 “Alternative Methodsfor Control of MicrobiologicalQuality”3 is now in force. Bothregional compendia recognized theneed to provide more appropriate

definitions to the accepted valida-tion criteria of accuracy, precision,limit of quantification, etc. This

was required as it was recognizedearly on that the established terms,

while appropriate for chemistry, were unworkable in the validationof microbiological assays due tothe larger degree of variability inthe system.3,4 While these “valida-tion guides” are useful, they

assume that the new technology is

in fact new and different from thecompendial methods.

There is a need to distinguishbetween “automated compendial”methods and “alternative” tests, asthey require different validationapproaches. Automated compen-dial tests differ from alternativemicrobiological tests in that theautomated tests are based on thesame methods and principles and

measure the same targets as themanual compendial tests. Alterna-tive tests, on the other hand, usedistinct methods and principlesand measure distinct targets, suchas ATP bioluminescence, “fluores-cent events,” etc., compared tocompendial tests.

It is also worth mentioning thatthe compendial “method” underreview may not actually be thetitle of the USP chapter. Forexample, the sterility test can bedescribed as two discrete steps:

1) Filtration of the sample

2) Examination of the filter forthe presence of viable cells.

A “rapid” sterility test willprobably have the same designas the compendial test (nowharmonized)—20 units of product

will be filtered, and the filter willbe assayed for viable cells.6 The“rapid” part only comes in as youspecify the method used to assayfor viable cells. Similarly, many quantitative assays do not differsignificantly from the compendialmethod except in the manner ofdetermining the number of cellspresent. Here there may be moreof a concern. The compendialmethod for enumeration is to grow colonies on or in an agar ➤

Compendial Requirements for Automated Microbiological Method Validation:The Role of USP Chapter <16> “Automated Methods of Analysis” and the Proposed Chapter

<1058> “Analytical Instrument Qualification”David Jones, Genomic Profiling Systems, and Scott Sutton, Vectech Pharmaceutical Consultants



PDA Letter • June 2006

24

surface. The colony forming unit(CFU) may arise from one cellor several thousand; it becomes

visible only after there are severaltens of millions in the colony afterreplication.

Alternate methods of enumeration

that are not based on the CFU arefundamentally different from thecompendial method of enumera-tion used in the microbial limitstests, the antimicrobial efficacytest and others. For example, the

AES Chemunex ScanRDI methodmeasures the numbers of cellsshowing esterase enzymaticactivity rather than the number ofcolony forming units—the quantitymeasured by the compendialmethods. Consequently, the targetsmeasured by the ScanRDI systemcan be very different than thosemeasured by the compendialtests, since not all of cells withesterase activity can replicate inthe presence of microbiologicalmedia7.

Automated compendial tests differfrom the manual compendialtests only in that some manipula-

tions and/or detection steps areautomated. For example, colonycounting by GPS’ Growth DirectSystem8 and the QCount fromSpiral Biotech uses the samemethod principles (growth ofcolonies on an agar surface) andmeasures the same colonies as dothe tests described in several USPchapters. Both the manual andautomated approaches enumeratecolonies derived from microbesthat can replicate on a mediasupport. The automated system,however, uses digital imaging todetect the colonies, in contrastto the manual method in whichcolonies are detected by eye.The automated imaging is morereproducible and allows fasterenumeration times.

For alternative tests, validationmust be concerned with demon-

strating that measuring differenttargets leads to equivalent or betterresults compared to the compen-dial methodology. However,USP <16> argues persuasivelythat an automated test need onlydemonstrate accuracy and preci-

sion. If we allow for the strategyand definitions in the proposedUSP Chapter <1223>, applicationof the approach embodied in USP<16> for validation of automatedmethods in microbiology shouldbe appropriate. In this approach,once the equipment is qualified,the method need demonstrate onlyaccuracy and precision equivalentto the compendial method.

This does bring up equipmentqualification as a concern. The2005 Pharmacopeial Preview forthe proposed USP chapter <1058>“Analytical Instrument Qualifica-tion” states:1

Good Manufacturing Practices(GMP) regulations requirecompanies to establish proce-dures ensuring the fitness for useof instruments that generate data supporting regulated product

testing. However, GMP regula-tions do not provide definitive guidance for the qualification ofanalytical instruments.

The chapter’s goals are described:

This chapter covers the initial part of the data quality acquisi-tion process (qualification,validation, and verification),defines the roles and responsibili-ties of those associated with an

instrument’s qualification, andestablishes the essential param-eters for performing instrumentqualification and a commonterminology.

In response to public concerns,USP published a revised draft

which presents the opportunity toaccept the system suitability testas proof of suitable performancefor the PQ portion of the quali-

fication.9 This chapter is beingfinalized for publication. Oncefinalized, it will serve not onlyfor automated microbiologicalmethods, but all equipment qualifi-cation studies.

A major concern with acceptance

of alternate microbiologicalmethods is uncertainty over valida-tion and the associated costs.However, the opportunities forthese methods to streamline testingis enormous.10,11 Clearly thedifferent types of alternate micro-biological methods have differingdegrees of risk associated withthem and should have differing

validation burdens. Many of theseautomated technologies clearly fallin the same philosophical categoryas was envisioned by USP in thecreation of a dedicated chapterdescribing the validation of anautomated, rather than an alterna-tive, method.

References:1 U.S. Pharmacopeia, “<1058> Analytical Instrument Qualification,” Pharmacopeial Forum, vol. 31, no.1 (2005), pp. 233-243.

2 U.S. Pharmacopeia, “<1223> Valida-tion of Alternative MicrobiologicalMethods,” Pharmacopeial Forum,

vol. 31, no. 5 (2005), pp. 1475-1486.3 European Pharmacopoeia, “5.1.6 Alternative Methods for Control ofMicrobiological Quality,” PharmEu-

ropa, Suppl. 5.5, pp 4131-4142.4 Sutton, S.V.W., “Validation of Alterna-

tive Microbiology Methodsfor Product Testing: Quantitative and

Qualitative Assays,” Pharmaceutical

Technoogy, vol. 29, no. 4 (2005), pp

118-122.5 Knapp, J.E., et al,, “Developing an

Information Chapter in the USPto Demonstrate Equivalency inMicrobiological Methods,” American

Pharmaceutical Review, vol. 5, no. 2(2002), pp. 14-19.

6 Moldenhauer, J. and S.V.W. Sutton,“Towards an Improved SterilityTest,” PDA Journal of Pharmaceuti-

cal Science and Technology, vol. 58,no. 6 (2004), pp. 284-286.

continued on page 2



PDA Letter • June 2006

26

7 Yvon, P., “Viability-based TechnologiSolid-phase Cytometry Using ChemuScanRDI,” Encyclopedia of Rapid Mic

biological Methods, vol. 2, edited by

M.J. Miller, pp 291-315 (PDA and DHPublishing, LLC, 2005).

8 London, R., et al., “The Growth DirecTest: A Rapid, Non-destructive Automed System for Microbial Enumeration Encyclopedia of Rapid Microbiologic

Methods, vol. 2, edited by M.J. Miller103-135 (PDA and DHI Publishing, L2005).

9 U.S. Pharmacopeia, “<1058> AnalyticInstrument Qualification,” Pharma-

copeial Forum, vol. 32, no. 2 (2006)pp:595-605.

10 Hussong, D. and R. Mello, “AlternatiMicrobiology Methods and Pharma-ceutical Quality Control,” American

Pharmaceutical Review. vol. 9, no. 1(2006), pp. 62-68.

11 Cundell, A.M., “Opportunities for RaMicrobial Methods,” European Pharm

ceutical Review, vol. 1 (2006), pp. 64

Next in the process was creationand review of a list of potentialmaterials of construction for thejacket and internal sealingsurfaces. Nine potential materials

in ten common chemicals weretested for hardness, change inmass, total organic carbon (TOC)extractables, nonvolatile residue(NVR), and small molecule clear-ance. By individually testing allmaterials, the team was able todetermine which provided the bestcombination of cleanliness andperformance.

The original list of ten items was

trimmed to three that met allacceptance criteria. These threematerials were then introducedin alpha product samples andsubjected to specific product tests.

As a result, the team selected thetwo materials that not only metall acceptance criteria but alsomaximized performance.

Pellicon® 3 then moved intothe detailed development stage.

Manufacturing and developmentengineers, under the guidanceof quality engineers, conductedexperiments to help understandthe contributions and interactionsof process variables on the processand final product. One projectrequirement was to identify criticalprocess parameters. The teamconducted numerous designs ofexperiment (DOE) on isolatedprocess steps to simplify the

development process.One basic building block for aPellicon® 3 device is a membranepacket consisting of a permeatescreen sandwiched between twomembranes. Membrane packetsare then separated by feed screensand stacked until the correctmembrane area is achieved.To increase throughput whileimproving quality and cleanli-

ness, the project team designedan automated packet assemblymachine (APAM) that is fed rollsof membrane and precut screen,

which are converted into finished

packets. The APAM producespackets that are then in-line andon-line tested before roboticallybeing stacked.

In determining the ideal machineoperating conditions, the teamconducted several DOEs to under-stand the impact of all process

variables employed in manufactur-ing the packets.

For example, experiments weredeveloped to measure the impactof each individual variable, as

well as combinations of multiple variables, on selected outputs suchas packet integrity or thickness(Figure 1, p. 28). Three critical heatsealing parameters were identified.This understanding allowed theengineers to focus on the mostcritical parameters and to establishappropriate operating specifica-

tions that result in a repeatableprocess that delivers packets ofknown performance characteristics(Figure 2, p. 28).

The knowledge gained from theDOEs enabled the proactive analy-sis of critical process parametersand will help in future develop-ment projects. Manufacturingengineers will continue to collectdata and increase their understand-ing of the process. These steps will

further reduce product variabilityby enabling modifications to thecontrol methods and/or limits.

Delivering Quality

The final cassette manufacturingprocesses include design andmanufacture to cGMP standards.These include cleaner design andmanufacturing environments, use

continued on page 28

A Supplier Approach to Ensuring Process and Product Quality,

continued from page 20

Compendial Requirements for Automa

Microbiological Method Validation,

continued from 24

About the AuthorsDavid Jones, PhD, is the Director of

Technical Services at Genomic ProfilinSystems. He has many years experien

with rapid microbiology methods,including time at Wyeth Biopharma

were he helped evaluate and validaterapid methods and new technologiesimprove laboratory efficiencies.

Scott Sutton, PhD, has had over 20 years of industrial experience withcompanies such as Bausch and Lomband Alcon Laboratories before joining

Vectech Pharmaceutical Consultants in

2004. Among his various affiliations, DSutton operates an information sourcethe internet—The Microbiology Netw(http://www.microbiol.org) that provservices to microbiology related user’groups—and supports the PMFList, ane-mail list devoted to pharmaceuticalmicrobiology (www.microbiol.org/pmflist_info.htm), and the Pharmaceucal Stability Discussion Group (www.

microbiol.org/psdglist_info.htm).

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Compendial Requirements for Automated Microbiological Method Validation